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Molecular & Cellular Oncology

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Mirror image phosphoinositides regulateautophagy

Mariella Vicinanza & David C Rubinsztein

To cite this article: Mariella Vicinanza & David C Rubinsztein (2016) Mirror imagephosphoinositides regulate autophagy, Molecular & Cellular Oncology, 3:2, e1019974, DOI:10.1080/23723556.2015.1019974

To link to this article: http://dx.doi.org/10.1080/23723556.2015.1019974

© 2016 The Author(s). Published withlicense by Taylor & Francis© MariellaVicinanza and David C Rubinsztein

Accepted author version posted online: 26Feb 2015.Published online: 26 Feb 2015.

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Mirror image phosphoinositidesregulate autophagy

Mariella Vicinanza and David C Rubinsztein*

Department of Medical Genetics; Cambridge Institute for Medical Research; Cambridge, UK

Keywords: autophagym, phosphoinositide, VPS34, PI(5)P

Autophagosome formation is stimulated by canonical VPS34-dependent formation of phosphatidylinositol3-phosphate [PI(3)P], which recruits effectors such as WIPI2. However, non-canonical VPS34-independent autophagyhas also been proposed. We recently described that PI(5)P regulates autophagosome biogenesis, recruits WIPI2, andrescues autophagy in VPS34-inactivated cells. These alternative autophagy-initiating pathways reveal new druggabletargets for treating neurodegeneration and cancer.

Phosphoinositides (PIs) are formed byphosphorylation of the inositol headgroupof phosphatidylinositol (Fig. 1A). Thecombined activities of multiple phosphoi-nositide kinases and phosphatases enableinterconvertibility and rapid local changesin PI concentrations. When clustered, PIscreate a cytosol-facing platform that ena-bles the binding of machineries that regu-late membrane deformation, and PIs canalso influence the geometry of the mem-brane due to their inverted cone shape.Since local PI concentrations respond tonutrient availability and cell stress theycan integrate membrane trafficking eventswithin other functional modules, such assignaling and energy control.

Autophagy is a bulk degradation pro-cess that delivers cytoplasmic materials(long-lived proteins and damaged organ-elles) to lysosomes, and can be induced byamino acid starvation, growth factor with-drawal, and low cellular energy levels. Thefirst morphologically recognizable precur-sor of this process is a flattened double-membraned sac-like structure called aphagophore, whose edges elongate andfuse while engulfing a small portion of thecytoplasm to become an autophagosome(Fig. 1B). Autophagosomes fuse withlysosomes to form autolysosomes, whosecontents can be degraded by the lysosomal

hydrolases. Autophagy impacts the patho-genesis of diverse diseases, including neu-rodegenerative conditions, cancers, andinfectious diseases.1

Autophagy has been classically consid-ered to be dependent on phosphatidylino-sitol 3-phosphate [PI(3)P], since geneticstudies in yeast and flies suggested that PI(3)P production by the class III phosphati-dylinositol 3-kinase (VPS34) is requiredfor autophagosome formation, for severalproteins associated with autophagosomebiogenesis (double FYVE-containingprotein 1 [DFCP1] and WD-repeat pro-tein interacting with phosphoinositide[WIPI1-2]),2 and for other components ofthe pathway.3 However, recent studies havealso suggested the existence of VPS34-inde-pendent autophagy.4For instance, autopha-gosomes form in T lymphocytes andsensory neurons from Vps34¡/¡ mice5 andin glucose-starved or resveratrol-treatedcells incubated with the VPS34 inhibitorwortmannin.4, 6 These phenomena may beattributed to VPS34-independent source(s)of PI(3)P,7 or to other phosphoinositideswith similar properties to PI(3)P in theautophagy context.

The structure of PI(5)P is related tothat of PI(3)P by rotation of the inositolring that interconverts the position of the3- and 5-phosphoryl groups, such that

their charges can occupy equivalent posi-tions (Fig. 1A). Although PI(5)P has beenimplicated in growth factor signalingpathways, chromatin organization, bacte-rial invasion, and cytoskeletal remodel-ing,8 our study is the first report of PI(5)Pas an autophagy regulator.9 This is com-patible with previous observations of simi-lar binding of PI(3)P and PI(5)P to WIPIproteins.10

PI(5)P biosynthesis is regulated by thetype III PtdInsP 5-kinase PIKfyve, eitherby direct phosphorylation of PI or via for-mation of PI(3,5)P2, which is then trans-formed into PI(5)P by 3-phosphatases ofthe myotubularin family (MTMRs)8

(Fig. 1A). The major route for PI(5)Premoval is attributed to type II PI5PKkinases (phosphatidylinositol 5-phosphate4-kinases, PI5P4K)8 (Fig. 1A). We foundthat increasing cellular PI(5)P levels (bythe addition of exogenous PI(5)P orsilencing of the PI5P4-kinases) enhancedautophagosome formation and autopha-gosome and autolysosome numbers,whereas depleting PI(5)P (via the PIKfyveinhibitor YM-201636 or by silencing ofPIKfyve or MTMR3) decreased the num-bers of autophagosome precursors andcompleted autophagosomes. Interestingly,PI(5)P (and its precursor PI(3,5)P2)localized on nascent and completed

© Mariella Vicinanza and David C Rubinsztein*Correspondence to: David C Rubinsztein; Email: dcr1000@cam.ac.ukSubmitted: 02/05/2015; Revised: 02/10/2015; Accepted: 02/11/2015http://dx.doi.org/10.1080/23723556.2015.1019974

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s)have been asserted.

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Molecular & Cellular Oncology 3:2, e1019974; March 2016; Published with license by Taylor & Francis Group, LLCAUTHOR'S VIEW

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autophagosomes and the kinases acting onPI(5)P localized on autophagosomes(Fig. 1B) under different type of starva-tion conditions.

PI(5)P resembles PI(3)P as a regulatorof autophagosome biogenesis. Acutedepletion of PI(3)P using wortmannincompletely ablated the presence of WIPI2and DFCP1 vesicles and inhibitedATG5-12 conjugation and autophago-some formation. PI(5)P regulates autoph-agy even when VPS34 is inhibited, sincethe impaired autophagosome formationcaused by wortmannin or VPS34 silencingwas rescued by elevating PI(5)P levels.

Our observations that PI(5)P binds toWIPI2, affects PI(3)P-related phenotypessuch as ATG12-5 conjugation, and canrescue autophagy in wortmannin-treatedHBSS-starved cells, argues that it can serveas an “alternative” to PI(3)P. Indeed, wefound that PI(3)P and PI(5)P can mutu-ally compete for binding to the PI(3)Pautophagy effector WIPI2 in vitro. Wepropose that PI(5)P might account for thepreviously mysterious phenomenon ofPI(3)P-independent, non-canonicalautophagy. Since PI(5)P could sustainautophagy in VPS34-inactive cells, we fur-ther confirmed that PI(5)P was involved

in glucose starvation- or resveratrol-induced autophagy, where PI(3)P isdispensable. Indeed, glucose starvation-induced autophagy appeared to be depen-dent on PI(5)P and not PI(3)P.

Our analysis reveals a hitherto unknownfunctional interplay between PIKfyve andPI5P4Ks in controlling PI(5)P levels in thecontext of autophagy and will pave the wayfor further studies of this exciting lipid inphysiological cellular processes, includingneurodegeneration and cancer. This mayhave therapeutic potential, because PIKfyveand PI5P4K enzymes are druggable targetsand our data indicate that suppression of

Figure 1. PI(5)P regulates autophagosome biogenesis. (A) Schematic representation of pathways for PI(5)P synthesis/turnover and enzymes involved inthese pathways. PI5P4K, type II phosphatidylinositol 5-phosphate 4-kinase; PIKfyve, type III phosphatidylinositol 5-phosphate 5-kinase; VPS34, class IIIphosphatidylinositol 3-kinase; PIK3C2A, class II phosphatidylinositol 3-kinase; MTMR, myotubularin related 3-phosphatase. (B) PI(5)P (red) is found onpre-phagophore structures, phagophores, and autophagosomes, and stabilizes membrane-bound WIPI2 on autophagic precursors. WIPI2 bindingrecruits the ATG16L1-5-12 complex, which enables phagophore biogenesis and subsequent LC3 lipidation and autophagic substrate degradation. PI(5)Pmetabolism on autophagosomes seems to be highly dependent on PIKfyve and PI5P4K activities. Importantly, although we found that PI(3)P (green)was required for amino acid starvation-induced autophagy, PI(5)P synthesis was essential for autophagy activation by both amino acid and glucosedepletion. Thus, we have identified PI(5)P as a new master regulator of glucose starvation-induced autophagy through a PI(3)P-independent autophagypathway.

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PI5P4K activity increased the clearance ofautophagic substrates associated with neu-rodegenerative disease. Autophagy induc-tion via other signaling effectors has benefitsin a wide range of cell and animal models ofneurodegenerative diseases caused by aggre-gate-prone intracytoplasmic proteins, likeHuntington’s and Parkinson’s disease.Thus, PI5P4K inhibition may provide atractable therapeutic target for such condi-tions. On the other hand, understanding

the enzymes that regulate autophagy via PI(5)P may also enable inhibition of the path-way in cancers. This may be relevant inmet-astatic cancers where autophagyupregulation induced by theWarburg effectmight contribute to tumor survival.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest weredisclosed.

Funding

We are grateful for funding froma Wellcome Trust Principal ResearchFellowship (DCR) (095317/Z/11/Z), aWellcome Trust Strategic Award(100140/Z/12/Z), the NIHR Biomedi-cal Research Center in Dementia atAddenbrooke’s Hospital, and an MRCConfidence in Concepts grant (DCR).

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